The class type I scavenger receptor (SR-BI) is the high density lipoprotein (HDL) receptor that regulates HDL- cholesterol metabolism and is directly linked to the ability of HDL to be athero-protective. The long-term objective of our research is to understand the function of SR-BI in the delivery of cholesteryl ester (CE) from HDL to the liver for cholesterol disposal. New insight into how SR-BI mediates the efficiency of HDL-CE delivery is key to developing methods for prevention of cardiovascular disease. This proposal consists of three primary objectives that will evaluate how the structural organization of SR-BI at the plasma membrane and the proper alignment of SR-BI with HDL mediate enhanced cholesterol flux to the liver. Aim 1 will determine the physiological organization and relevance of the SR-BI oligomer in vivo. Goal 1 will use bimolecular fluorescence complementation coupled with fluorescence resonance energy transfer spectroscopy to confirm the presence of SR-BI oligomers in live cells and monitor changes in oligomer formation upon ligand engagement. In Goal 2, the physiological relevance of SR-BI oligomerization in reverse cholesterol transport will be assessed following adenoviral-mediated expression of oligomerization-defective mutant SR-BI receptors in SR-BI knock-out mice. Aim 2 is designed to examine the molecular determinants for productive complex formation (i.e. proper alignment) between HDL and SR-BI that promote selective uptake of HDL-CE. In Goal 1, a series of SR-BI/CD36 chimeras will be designed to identify regions within the extracellular domain of SR-BI that are crucial for HDL-CE selective uptake and vital for productive complex formation. In Goal 2, the combination of site-specific ligand-directed crosslinking and mass spectrometry will be used to map sites of interaction between SR-BI and HDL. Aim 3 will explore how the conformation of the extracellular domain of SR-BI impacts lipid transfer from HDL to the plasma membrane. Goal 1 will use tryptophan quenching to test the hypothesis that hydrophobic regions of SR-BI are required to interact with the plasma membrane and/or ligand to facilitate efficient lipid transfer and cholesterol flux. Goal 2 will determine the role of extracellular cysteine residues in SR-BI function and experiments are designed to identify intra- and intermolecular disulfide bonding patterns. Together, these studies will improve our understanding of how SR-BI mediates the efficiency of HDL-CE selective uptake and will shed new insights into cholesterol metabolism and protection against atherosclerosis. PUBLIC HEALTH RELEVANCE: Heart disease kills more Americans each year than all cancers combined. Our research is designed to understand how we can improve cholesterol removal from the body and lower plasma cholesterol levels. Our findings will help identify new strategies for treating heart disease and other related complications.